Folic acid and creatine improve the firmness of human skin in vivo Frank Fischer, PhD, * Volker Achterberg, PhD, * Annette Ma ¨ rz, PhD, Stefan Puschmann, BE, Christian-Dennis Rahn, PhD, Vivien Lutz, MSc, Andrea Kru ¨ ger, Laboratory Technician, Helge Schwengler, Laboratory Technician, So ¨ ren Jaspers, Graduate Engineer of Medical Technology, Urte Koop, Graduate Engineer of Medical Technology, Thomas Blatt, PhD, Horst Wenck, PhD, & Stefan Gallinat, PhD Research & Development, Skin Research, Beiersdorf AG, Hamburg, Germany Summary Background The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. Aims Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age-dependent reduction in the amount of collagen. Patients ⁄ Methods Topical application of a commercially available formulation contain- ing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast-populated collagen gels were carried out. Results The underlying structural changes in the collagen network of young and aged sun-exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid- and creatine-containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast-populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved col- lagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. Conclusions Employing MPLSM, dermal changes occurring in photo-aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age-dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives. Keywords: collagen, creatine, folic acid, multiphoton laser scanning microscopy, skin aging, second harmonic generation Introduction The firmness of human skin is to a major extent determined by the dermis. This dermal structural support is primarily dependent on the composition of the dermal extracellular matrix (ECM) that is mainly composed of collagens. Correspondence: Volker Achterberg, Beiersdorf AG, Research & Development, Department of Skin Biology & Skin Structure, Bf. 510, Unnastrasse 48, 20245 Hamburg, Germany. E-mail: volker.achterberg@ beiersdorf.com * The first two authors contributed equally to this work. Accepted for publication November 14, 2010 Original Contribution Ó 2011 Wiley Periodicals, Inc. • Journal of Cosmetic Dermatology, 10, 15–23 15